Stephen J Russell
Stephen J Russell is at the Mayo Clinic and Foundation, Guggenheim 18, 200 First Street SW, Rochester, MN 55905, USA. e-mail: sjr@mayo.edu
Nanoparticles that combine features of viral and nonviral vectors show promise for targeted gene delivery.
A contest is currently underway between synthetic nonviral vectors and recombinant viral vectors to determine supremacy in gene therapy applications[1]. Viruses have a considerable advantage, with a head start of several hundred thousand years in perfecting the art of gene delivery. Humans are very fast learners, however, and the gap between viral and nonviral vectors is rapidly closing, as demonstrated on p. 885 of this issue. In their paper, Yamada et al.[2] used a yeast expression system to generate hollow lipid nanoparticles displaying the hepatitis B virus (HBV) attachment protein, and filled them with plasmid DNA (or protein) by electroporation. The resulting DNA-loaded vector particles are able to mediate efficient targeted gene delivery to cultured human hepatocytes and to human hepatoma cells both in vitro and in vivo. Moreover, the target cell specificity of the vectors can be redirected by engineering a cell-targeting domain into the nanoparticle coat.
The story of these remarkable new nanomachines began over a decade ago when the same group of researchers was aggressively pursuing the goal of generating an improved HBV vaccine[3]. At that time, the authors expressed the HBV envelope L proteins in recombinant yeast cells and harvested L particles, each comprising about 110 molecules of the L protein embedded in a phospholipid vesicle derived from the yeast endoplasmic reticulum. The L proteins are displayed on the surface of the L particles where, as in HBV, they function as specific ligands for human hepatocytes[4]. Physical characterization of the L particles revealed that they are roughly spherical and relatively large, with diameters ranging from 50 to 500 nm[5].
Given that L particles can bind and fuse with hepatocytes and have substantial internal capacity for foreign cargo, the idea of loading them with DNA and using them for liver gene transfer was a logical development. But how to get the DNA inside the preformed particles without destroying them? Viruses do not offer clues to a convenient solution as they package their nucleic acids during, not after, the process of particle assembly[6]. In addition, synthetic gene transfer vectors are made by premixing unassembled component parts (e.g., condensing plasmid DNA with cationic lipids) or alternatively by coating the DNA onto preformed nanoparticles[7] (see Fig. 1).
Yamada et al. took the ingenious approach of incorporating genes into the L particles by electroporation. Surprisingly, the electroporation procedure did not damage or alter the physical characteristics of the L particles, which were able deliver their new genetic payloads with high specificity to cultured human hepatocytes. Moreover, the specificity of gene delivery could be manipulated by generating L particles expressing a new cell binding domain (human epidermal growth factor) genetically fused to the N terminus of the L protein.
One of the major drawbacks of most currently available vectors, whether viral or nonviral, is lack of specificity, leading to unwanted gene transfer to nontarget tissues[8]. L protein nanoparticles therefore have the potential to become useful agents for targeted delivery of genes, proteins or other electroporatable drugs to human liver. One can also envisage an array of targeted L protein nanoparticles suitable for targeted gene or drug delivery to a variety of tissues. An additional potential advantage of this new technology is that, in contrast to conventional DNA nanoparticles, which enter their target cells by endosomal disruption[7], the L protein nanoparticles deliver their payload by fusing with the target cell membrane, a relatively nontoxic entry strategy.
Unfortunately, L protein nanoparticles are not without their problems. One significant concern is that, as originally intended, the L particles are highly immunogenic, provoking antibody responses against all three HBV coat proteins[5]. And, with the successful global deployment of effective HBV vaccines, a large proportion of the world's population now has antibodies to HBV[9]. Thus, as the authors discuss in their paper, unless the particles can be further engineered to circumvent these antibodies, there is a concern that they may be rapidly sequestered and neutralized before they reach their target sites. In addition, further work will be required to increase the transfection efficiency of the L protein nanoparticles as it took a high multiplicity of infection (104–105 particles per cell) to achieve a high efficiency transduction of HepG[2] hepatocellular carcinoma cells.
Overall, the development of L protein nanoparticles for gene and drug delivery offers an entirely new and promising class of synthetic delivery vehicle, created by merging viral with nonviral components. Thus, the efficient, hepatocyte-specific entry strategy of HBV has been combined with the reassuring safety features and high cargo capacity of nonviral vectors. Electroporation of DNA into preformed nanoparticles derived by expressing viral proteins in yeast was the key to this new technology, a development that will doubtless pave the way for numerous additional hybrid viral-nonviral vectors.
Perhaps, with perfection, these synthetic nanomachines will become increasingly difficult to discriminate from viruses. In any event, the battle of the vectors is getting very interesting.
REFERENCES
doi:10.1038/nbt0803-872
August 2003 Volume 21 Number 8 pp 872 - 873
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